Antisense compositions for detecting and inhibiting telomerase reverse transcriptase

ABSTRACT

The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional of U.S. patent application Ser. No. 09/052,919, filed Mar. 31, 1998. The present application is a continuation-in-part of U.S. patent application Ser. No. 08/974,549, filed Nov. 19, 1997, and a continuation-in-part of U.S. patent application Ser. No. 08/974,584, filed Nov. 19, 1997, both of which are continuation-in-part applications of U.S. patent application Ser. No. 08/915,503, U.S. patent application Ser. No. 08/912,951, and U.S. patent application Ser. No. 08/911,312, all filed Aug. 14, 1997, all three of which are continuation-in-part applications of U.S. patent application Ser. No. 08/854,050, filed May 9, 1997, which is a continuation-in-part application of U.S. patent application Ser. No. 08/851,843, filed May 6, 1997, which is a continuation-in-part application of U.S. patent application Ser. No. 08/846,017, filed Apr. 25, 1997, which is a continuation-in-part application of U.S. patent application Ser. No. 08/844,419, filed Apr. 18, 1996, which is a continuation-in-part application of U.S. patent application Ser. No. 08/724,643, filed Oct. 1, 1996. This application is also a continuation-in-part of Patent Convention Treaty Patent Application Serial No.: PCT/US97/17885 and to Patent Convention Treaty Patent Application Serial No.: PCT/US97/17618, both filed on Oct. 1, 1997. Each of the aforementioned applications is explicitly incorporated herein by reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] The present invention provides TRT antisense oligonucleotides, methods of detecting TRT, methods of diagnosing telomerase-related conditions, methods of diagnosing and providing a prognosis for cancer, and methods of treating telomerase-related conditions, including cancer, with TRT antisense oligonucleotides.

BACKGROUND OF THE INVENTION

[0003] The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section should not be construed as an admission of prior invention.

[0004] It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972, Nature New Biol., 239:197; Olovnikov, 1973, J. Theor. Biol., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5′ to 3′. When the RNA bound at the extreme 5′ ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence or aging of normal human somatic cells in vitro and in vivo. The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity), or the ability of a cell to escape senescence, i.e., to become immortal. The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomeric DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750).

[0005] Consistent with the relationship of telomeres and telomerase to the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBO J. 11:1921; Counter et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416). Human telomerase is thus an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer.

SUMMARY OF THE INVENTION

[0006] The present invention provides TRT antisense polynucleotides, which are useful for detecting, diagnosing, and treating telomerase-related conditions.

[0007] In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence that is substantially complementary or substantially identical to a contiguous sequence of an hTRT nucleic acid that has the nucleotide sequence of FIG. 1.

[0008] In one aspect, the present invention provides an isolated, synthetic, substantially pure, or recombinant polynucleotide having a sequence that is at least about ten nucleotides in length to at least about 100 nucleotides in length. This polynucleotide comprises a sequence exactly complementary or identical to a contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 2.

[0009] In one embodiment, the hTRT polynucleotide comprises a sequence that is exactly complementary or identical to a contiguous sequence of an hTRT nucleic acid having the nucleotide sequence of FIG. 1.

[0010] In one embodiment, the polynucleotide is a DNA or an RNA. In one embodiment, the polynucleotide comprises one or more non-naturally occurring, synthetic nucleotides.

[0011] In one embodiment, the polynucleotide is identical to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1. In one embodiment, the polynucleotide is exactly complementary to said contiguous sequence of a nucleic acid encoding the hTRT protein of FIG. 1.

[0012] In one embodiment, the polynucleotide is an antisense polynucleotide. In one embodiment, the polynucleotide is at least about 20 nucleotides in length to at least about 50 nucleotides in length.

[0013] In one embodiment, the polynucleotide inhibits telomerase activity by at least about 50% in transformed cells ex vivo, as compared to control cells that are not treated with the polynucleotide. In one embodiment, the polynucleotide inhibits telomerase expression by at least about 50% in vitro, as compared to control expression reactions that lack the polynucleotide. In one embodiment, the polynucleotide is selected from the group consisting of PS-ODN number 3, 4, 7, 8, 16, 21, 25, 26, 27, 28, 29, 33, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 62, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 80, 81, 82, 83, 84, 85, 86, 87, 88, 93, 94, 96, 100, 112, 114, 130, 143, 144, 151, 152, 201, 202, 203, 208, 209, 210, 211, 212, 213, 230, 237, and 241.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 presents the nucleotide sequence of a cDNA encoding a naturally occurring human telomerase reverse transcriptase (hTRT) protein.

[0015]FIG. 2 presents the amino acid sequence of a naturally occurring, 1132-residue human telomerase reverse transcriptase (hTRT) protein.

[0016]FIG. 3 shows inhibition of hTRT expression in vitro by hTRT sequence-specific antisense phosphorothioate oligonucleotides (PS-ODN). Each bar in the graph represents the in vitro inhibitory activity of a specific oligonucleotide, numbered starting with PS-ODN #1. The PS-ODN are a series of 30-mers that span the hTRT mRNA and are offset one from the next by fifteen nucleotides. For example, ODN #1 corresponds to positions 16-35 of hTRT and is TCCCACGTGCGCAGCAGGACGCAGCGCTGC. ODN 2 corresponds to positions 31-60 and is GGCATCGCGGGGGTGGCCGGGGCCAGGGCT, and so one to the end of the RNA (see the cDNA sequence of FIG. 1, which represents an hTRT RNA sequence). The data are presented as a normalized percentage of the control with no added PS-ODN.

DETAILED DESCRIPTION

[0017] I. Introduction

[0018] Telomerase is a ribonucleoprotein complex (RNP) comprising an RNA component and a catalytic protein component. The catalytic protein component of human telomerase, hereinafter referred to as telomerase reverse transcriptase (“hTRT”), has been cloned, and protein, cDNA, and genomic sequences determined. See, e.g., Nakamura et al., 1997, Science 277:955, and copending U.S. patent applications Ser. Nos. 08/912,951 and 08/974,549. The sequence of a full-length native hTRT has been deposited in GenBank (Accession No. AF015950), and plasmid and phage vectors having hTRT coding sequences have been deposited with the American Type Culture Collection, Rockville, Maryland (accession numbers 209024, 209016, and 98505). The catalytic subunit protein of human telomerase has also been referred to as “hEST2” (Meyerson et al., 1997, Cell 90:785), “hTCS1” (Kilian et al., 1997, Hum. Mol. Genet. 6:2011), “TP2” (Harrington et al., 1997, Genes Dev. 11:3109), and “hTERT” (e.g., Greider, 1998, Curr. Biol. 8:R178-R181). The RNA component of human telomerase (hTR) has also been characterized (see U.S. Pat. No. 5,583,016).

[0019] Human TRT is of extraordinary interest and value because, inter alia, telomerase activity in human cells and other mammalian cells correlates with cell proliferative capacity, cell immortality, and the development of a neoplastic phenotype. hTRT antisense polynucleotides, including the exemplary polynucleotides described herein, hybridize to and/or amplify naturally occurring hTRT genes or RNA. Such oligonucleotides are thus useful for diagnostic or prognostic applications to telomerase related conditions, including cancer. The hTRT antisense polynucleotides of the invention are also useful as therapeutic agents, e.g., antisense oligonucleotides, ribozymes, or triplex compositions, for inhibition of telomerase expression and activity (e.g., telomerase catalytic activity, infra).

[0020] The invention thus provides antisense oligonucleotide reagents, which can be used to detect expression of hTRT or reduce expression and activity of hTRT gene products in vitro, ex vivo, or in vivo. Administration of the antisense reagents of the invention to a target cell results in reduced telomerase activity, and is particularly useful for treatment of diseases characterized by high telomerase activity (e.g., cancers). Detection and inhibition of hTRT expression can be performed in a cell or cell extracts from a human, a mammal, a vertebrate, or other eukaryote.

[0021] The antisense polynucleotides of the invention are characterized by their ability to specifically hybridize to naturally occurring and synthetic hTRT nucleic acids, e.g., the hTRT gene, including any upstream, flanking, noncoding, and transcriptional control elements, hTRT pre-mRNA, mRNA, cDNA and the like. The hTRT antisense polynucleotides of the invention are typically at least 7-10 nucleotides in length to typically more 20 nucleotides up to about 100 nucleotides in length, preferably approximately 30 nucleotides in length. Such antisense oligonucleotides are used to detect the presence of hTRT nucleic acid in a biological sample, for diagnosis and/or prognosis of telomerase related conditions, e.g., cancers of any of a wide variety of types, including solid tumors and leukemias, diseases of cell proliferation, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, disease of immune dysfunction, etc.

[0022] The antisense polynucleotides of the invention also can be used to inhibit telomerase expression in vitro, to inhibit telomerase expression and activity in cells ex vivo, and can be used in vivo as therapeutic agents for the treatment of telomerase-related conditions listed above, including cancers of a wide variety of types (see, e.g., exemplary cancers listed in U.S. patent application Ser. No. 08/974,549; and U.S. patent application Ser. No. 08/974,584). In one embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression at least by 50% in vitro (see, e.g., the antisense oligonucleotides of FIG. 3). In another embodiment of the invention, the antisense polynucleotides are 30 nucleotides in length, and have the ability to inhibit telomerase expression and activity at least 50% in transformed cells ex vivo (see, e.g., exemplary antisense hTRT oligonucleotides listed in Table 1).

[0023] II. Definitions

[0024] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0025] As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably. Use of the term “polynucleotide” includes oligonucleotides (i.e., short polynucleotides). This term also refers to deoxyribonucleotides, ribonucleotides, and naturally occurring variants, and can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogues or modified backbone residues or linkages), such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like, as described herein.

[0026] As used herein “oligonucleotides” or “oligomers” refer to a nucleic acid sequence of approximately 7 nucleotides or greater in length, and up to as many as approximately 100 nucleotides in length, which can be used as a primer, probe or amplimer. Oligonucleotides are often between about 10 and about 50 nucleotides in length, more often between about 14 and about 35 nucleotides, very often between about 15 and about 30 nucleotides, and the terms oligonucleotides or oligomers can also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., comprising nucleic acid analogues or modified backbone residues or linkages).

[0027] A polynucleotide “specifically hybridizes” or “specifically binds” to a target polynucleotide if the polynucleotide hybridizes to the target under stringent conditions. As used herein, “stringent hybridization conditions” or “stringency” refers to conditions in a range from about 5° C. to about 20° C. or 25° C. below the melting temperature (T_(m)) of the target sequence and a probe with exactly or nearly exactly complementarity to the target. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the T_(m) of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic Press, Inc.; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory hereinafter, “Sambrook”); and Current Protocols in Molecular Biology (Ausubel et al., eds. through and including the 1997 supplement), incorporated herein by reference). As indicated by standard references, a simple estimate of the T_(m) value may be calculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of T_(m). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, and the like), and the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art, e.g., Sambrook, supra and Ausubel et al. supra. Typically, stringent hybridization conditions are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 30° C. for short nucleic acids (e.g., 7 to 50 nucleotides) and at least about 60° C. for long nucleic acids (e.g., greater than 50 nucleotides). As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.

[0028] An “identical” polynucleotide refers to a polynucleotide that has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared. An “exactly complementary” polynucleotide refers to a polynucleotide whose complement has the same sequence as the reference nucleotide subsequence to which the polynucleotide is being compared.

[0029] A “substantially complementary” polynucleotide and a “substantially identical” polynucleotide have the ability to specifically hybridize to a reference gene, DNA, cDNA, or mRNA, e.g., the hTRT nucleotide sequence of FIG. 1 and its exact complement.

[0030] An “antisense” polynucleotide is a polynucleotide that is substantially complementary to a target polynucleotide and has the ability to specifically hybridize to the target polynucleotide.

[0031] A “telomerase-related condition” refers to a diseases and disease conditions in a patient and/or a cell, characterized by under- or over-expression of telomerase or hTRT gene products. In addition to cancer, which is characterized by over-expression of telomerase, such conditions include diseases of cell proliferation, e.g., hyperplasias, disease resulting from cell senescence (particularly diseases of aging), immunological disorders, infertility, etc.

[0032] As used herein, “isolated,” when referring to a molecule or composition, such as, for example, an oligonucleotide, means that the molecule or composition is separated from at least one other compound, such as other oligonucleotides or other contaminants with which it is associated in vivo or in its naturally occurring state or synthetic state. An isolated composition can also be substantially pure.

[0033] A “synthetic” oligonucleotide refers to a polynucleotide synthesized using in vitro chemical methods, e.g., by using a machine that synthesizes polynucleotides using the phosphodiester method, the diethylphosphoramidite method, the phosphotriester methods, the solid support method, and other methods known to those skilled in the art.

[0034] As used herein, “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide.

[0035] As used herein, the term “substantially pure,” or “substantially purified,” when referring to a composition comprising a specified reagent, such as an oligonucleotide, means that the specified reagent is at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the composition (not including, e.g., solvent or buffer). Thus, for example, an antisense oligonucleotide preparation that specifically binds an hTRT gene or mRNA is substantially purified.

[0036] “TRT” activity refers to one or more of the activities found in naturally-occurring full-length TRT proteins. These activities include “telomerase catalytic activity” (the ability to extend a DNA primer that functions as a telomerase substrate by adding a partial, one, or more than one repeat of a sequence, e.g., TTAGGG, encoded by a template nucleic acid, e.g., hTR), “telomerase conventional reverse transcriptase activity” (see Morin, 1997, supra, and Spence et al., 1995, Science 267:988); “nucleolytic activity” (see Morin, 1997, supra; Collins and Grieder, 1993, Genes and Development 7:1364; Joyce and Steitz, 1987, Trends Biochem. Sci. 12:288); “primer (telomere) binding activity” (see, Morin, 1997, supra; Collins et al., 1995, Cell 81:677; Harrington et al., 1995, J. Biol. Chem. 270:8893); “dNTP binding activity” (Morin, 1997, supra; Spence et al., supra); and “RNA (e.g., hTR) binding activity” (see Morin, 1997, supra; Harrington et al., 1997, Science 275:973; Collins et al., 1995, Cell 81:677).

[0037] “TRT” refers to telomerase reverse transcriptase protein, and “hTRT” refers to human telomerase reverse transcriptase protein.

[0038] The term “hTRT” is intended to refer to alleles, conservatively modified variants, polymorphic variants, and interspecies homologues of hTRT encoded by nucleic acids that specifically hybridize to the hTRT nucleic acid sequence provided in FIG. 1.

[0039] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0040] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art (see, e.g., Creighton (1984) Proteins, W. H. Freeman and Company.

[0041] III. How to make antisense polynucleotides

[0042] As described herein, the present invention provides antisense polynucleotides, which have the ability to specifically hybridize to hTRT. Without intending to be limited to any particular mechanism, it is believed that antisense oligonucleotides bind to, and interfere with the translation of, the sense hTRT mRNA. Alternatively, the antisense molecule may render the hTRT mRNA susceptible to nuclease digestion, interfere with transcription, interfere with processing, localization or otherwise with RNA precursors (“pre-mRNA”), repress transcription of mRNA from the hTRT gene, or act through some other mechanism. However, the particular mechanism by which the antisense molecule reduces hTRT expression is not critical.

[0043] Generally, to assure specific hybridization, the antisense sequence is substantially complementary to the target hTRT mRNA sequence. In certain embodiments, the antisense sequence is exactly complementary to the target sequence. The antisense polynucleotides may also include, however, nucleotide substitutions, additions, deletions, transitions, transpositions, or modifications, or other nucleic acid sequences or non-nucleic acid moieties so long as specific binding to the relevant target sequence corresponding to hTRT RNA or its gene is retained as a functional property of the polynucleotide.

[0044] In one embodiment, the antisense sequence is complementary to relatively accessible sequences of the hTRT mRNA (e.g., relatively devoid of secondary structure). These sequences can be determined by analyzing predicted RNA secondary structures using, for example, the MFOLD program (Genetics Computer Group, Madison Wis.) and testing in vitro or in vivo as is known in the art. FIG. 3 and TAble 1 show examples of oligonucleotides that are useful in cells for antisense suppression of hTRT function and are capable of hybridizing to hTRT (i.e., are substantially complementary to hTRT). Another useful method for identifying effective antisense compositions uses combinatorial arrays of oligonucleotides (see, e.g., Milner et al., 1997, Nature Biotechnology 15:537).

[0045] A. Triplex-forming antisense polynucleotides

[0046] As one embodiment of the antisense molecules described herein, the present invention provides polynucleotides that bind to double-stranded or duplex hTRT nucleic acids (e.g., in a folded region of the hTRT RNA or in the hTRT gene), forming a triple helix-containing, or “triplex” nucleic acid. Triple helix formation results in inhibition of hTRT expression by, for example, preventing transcription of the hTRT gene, thus reducing or eliminating telomerase activity in a cell. Without intending to be bound by any particular mechanism, it is believed that triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules to occur.

[0047] Triplex oligo- and polynucleotides of the invention are constructed using the base-pairing rules of triple helix formation (see, e.g., Cheng et al., 1988, J. Biol. Chem. 263: 15110; Ferrin and Camerini-Otero, 1991, Science 354:1494; Ramdas et al., 1989, J. Biol. Chem. 264:17395; Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 9591; each of which is incorporated herein by reference) and the hTRT mRNA and/or gene sequence. Typically, the triplex-forming oligonucleotides of the invention comprise a specific sequence of from about 10 to at least about 25 nucleotides or longer “complementary” to a specific sequence in the hTRT RNA or gene (i.e., large enough to form a stable triple helix, but small enough, depending on the mode of delivery, to administer in vivo, if desired). In this context, “complementary” means able to form a stable triple helix. In one embodiment, oligonucleotides are designed to bind specifically to the regulatory regions of the hTRT gene (e.g., the hTRT 5′-flanking sequence, promoters, and enhancers) or to the transcription initiation site, (e.g., between −10 and +10 from the transcription initiation site). For a review of recent therapeutic advances using triplex DNA, see Gee et al., in Huber and Carr, 1994, Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N.Y. and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA 94:5854, which are both incorporated herein by reference.

[0048] B. Ribozymes

[0049] In another embodiment, the present invention provides ribozymes useful for inhibition of hTRT telomerase activity. The ribozymes of the invention bind and enzymatically cleave and inactivate hTRT mRNA. Useful ribozymes can comprise 5′-and 3′-terminal sequences complementary to the hTRT mRNA and can be engineered by one of skill on the basis of the hTRT mRNA sequence disclosed herein (see PCT publication WO 93/23572, supra). Ribozymes of the invention include those having characteristics of group I intron ribozymes (Cech, 1995, Biotechnology 13:323) and others of hammerhead ribozymes (Edgington, 1992, Biotechnology 10:256).

[0050] Ribozymes of the invention include those having cleavage sites such as GUA, GUU and GUC. Other optimum cleavage sites for ribozyme-mediated inhibition of telomerase activity in accordance with the present invention include those described in PCT publications WO 94/02595 and WO 93/23569, both incorporated herein by reference. Short RNA oligonucleotides between 15 and 20 ribonucleotides in length corresponding to the region of the target hTRT gene containing the cleavage site can be evaluated for secondary structural features that may render the oligonucleotide more desirable. The suitability of cleavage sites may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays, or by testing for in vitro ribozyme activity in accordance with standard procedures known in the art.

[0051] As described by Hu et al., PCT publication WO 94/03596, incorporated herein by reference, antisense and ribozyme functions can be combined in a single oligonucleotide. Moreover, ribozymes can comprise one or more modified nucleotides or modified linkages between nucleotides, as described above in conjunction with the description of illustrative antisense oligonucleotides of the invention.

[0052] C. Synthesis of antisense polynucleotides

[0053] The antisense nucleic acids (DNA, RNA, modified, analogues, and the like) can be made using any suitable method for producing a nucleic acid, such as the chemical synthesis and recombinant methods disclosed herein and known to one of skill in the art. In one embodiment, for example, antisense RNA molecules of the invention may be prepared by de novo chemical synthesis or by cloning. For example, an antisense RNA that hybridizes to hTRT mRNA can be made by inserting (ligating) an hTRT DNA sequence in reverse orientation operably linked to a promoter in a vector (e.g., plasmid). Provided that the promoter and, preferably termination and polyadenylation signals, are properly positioned, the strand of the inserted sequence corresponding to the noncoding strand will be transcribed and act as an antisense oligonucleotide of the invention.

[0054] The present invention also provides hTRT antisense polynucleotides (RNA, DNA or modified) that can be produced by direct chemical synthesis. Chemical synthesis is generally preferred for the production of oligonucleotides or for oligonucleotides and polynucleotides containing nonstandard nucleotides (e.g., probes, primers and antisense oligonucleotides). Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859 (1981); and the solid support method of U.S. Pat. No. 4,458,066.

[0055] Chemical synthesis typically produces a single stranded oligonucleotide, which may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase and an oligonucleotide primer using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is often limited to sequences of about 100 or 150 bases, longer sequences may be obtained by the ligation of shorter sequences or by more elaborate synthetic methods.

[0056] It will be appreciated that the hTRT polynucleotides and oligonucleotides of the invention can be made using nonstandard bases (e.g., other than adenine, cytidine, guanine, thymine, and uridine) or nonstandard backbone structures to provides desirable properties (e.g., increased nuclease-resistance, tighter-binding, stability or a desired T_(M)). Techniques for rendering oligonucleotides nuclease-resistant include those described in PCT publication WO 94/12633. A wide variety of useful modified oligonucleotides may be produced, including oligonucleotides having a peptide-nucleic acid (PNA) backbone (Nielsen et al., 1991, Science 254:1497) or incorporating 2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methyl phosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidates. Still other useful oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃, where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃ ; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a folate group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Folate, cholesterol or other groups that facilitate oligonucleotide uptake, such as lipid analogs, may be conjugated directly or via a linker at the 2′ position of any nucleoside or at the 3′ or 5′ position of the 3′-terminal or 5′-terminal nucleoside, respectively. One or more such conjugates may be used. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other embodiments may include at least one modified base form or “universal base” such as inosine, or inclusion of other nonstandard bases such as queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

[0057] The invention further provides oligonucleotides having backbone analogues such as phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, chiral-methyl phosphonates, nucleotides with short chain alkyl or cycloalkyl intersugar linkages, short chain heteroatomic or heterocyclic intersugar (“backbone”) linkages, or CH₂—NH—O—CH₂, CH₂—N(CH₃)—OCH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—P—O—CH₂), or mixtures of the same. Also useful are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506).

[0058] Useful references include Oligonucleotides and Analogues, A Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan et al., Jul. 9, 1993, J. Med. Chem. 36(14):1923-1937; Antisense Research and Applications (1993, CRC Press), in its entirety and specifically Chapter 15, by Sanghvi, entitled “Heterocyclic base modifications in nucleic acids and their applications in antisense oligonucleotides;” and Antisense Therapeutics, ed. Sudhir Agrawal (Humana Press, Totowa, N.J., 1996).

[0059] D. Labeled antisense oligonucleotides

[0060] It is often useful to label the antisense polynucleotides of the invention, for example, when the hTRT polynucleotides are to be used for detection of hTRT expression, and for diagnosis and prognosis of telomerase related conditions. The labels may be incorporated by any of a number of means well known to those of skill in the art. Suitable labels are any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²p, ³⁵S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, digoxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target. The label often generates a measurable signal, such as radioactivity, that can be used to quantitate the amount of bound detectable moiety.

[0061] The label can be incorporated in or attached to a polynucleotide either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety. For example, the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules.

[0062] IV. Exemplary antisense polynucleotides

[0063] A series of 30-mer antisense oligonucleotides, which span the entire hTRT sequence, are exemplary embodiments of the present invention (see FIG. 3). These oligonucleotides were systematically assayed for the ability to inhibit hTRT expression in vitro. The results of the experiment are presented in FIG. 3 (see also Example I). Any suitable series of hTRT antisense oligonucleotides can be tested in a similar fashion. For example, a series of 20-mer antisense oligonucleotides, offset one from the next by 10 nucleotides can be synthesized and tested in the same manner. A series of 25-mer, 35-mer, or 15-mer oligonucleotides can be examined in the same manner.

[0064] Selected oligonucleotides from the series of FIG. 3 were then tested ex vivo for their ability to inhibit hTRT expression in tumor cells (see Example II). The hTRT antisense oligonucleotides active for inhibiting telomerase activity ex vivo in tumor cells were than assayed for their long term cell culture effects on hTRT expression, telomerase activity, telomere dynamics, and cell proliferation (see Example II). The oligonucleotides of Table I represent exemplary oligonucleotides that inhibited telomerase activity ex vivo. TABLE I hTRT AS 30-mers: Telomerase Activity Relative to Untreated Cells PS- Position ODN# (3′-5′) 5′-AS sequence-3′ 3   31-60   GGCATCGCGGGGGTGGCCGGGGCCAGGGCT 4   46-75   CAGCGGGGAGCGCGCGGCATCGCGGGGGTG 7   91-120  AGCACCTCGCGGTAGTGGCTGCGCAGCAGG 8  106-135  AACGTGGCCAGCGGCAGCACCTCGCGGTAG 16  226-255  GCGGGGGGCGGCCGTGCGTCCCAGGGCACG 21  301-330  CCGCGCTCGCACAGCCTCTGCAGCACTCGG 25  361-390  GGGGGGCCCCCGCGGGCCCCGTCCAGCAGC 26  376-405  GTGGTGAAGGCCTCGGGGGGGCCCCCGCGG 27  391-420  TAGCTGCGCACGCTGGTGGTGAAGGCCTCG 28  406-435  ACCGTGTTGGGCAGGTAGCTGCGCACGCTG 29  421-450  CGCAGTGCGTCGGTCACCGTGTTGGGCAGG 33  481-510  AGGTGAACCAGCACGTCGTCGCCCACGCGG 40  586-615  GGGGGCCGGGCCTGAGTGGCAGCGCCGAGC 41  601-630  CCACTAGCGTGTGGCGGGGGCCGGGCCTGA 43  631-660  GCCCGTTCGCATCCCAGACGCCTTCGGGGT 44  646-675  ACGCTATGGTTCCAGGCCCGTTCGCATCCC 45  661-690  ACCCCGGCCTCCCTGACGCTATGGTTCCAG 46  676-705  GGCAGGCCCAGGGGGACCCCGGCCTCCCTG 47  691-720  CTCGCACCCGGGGCTGGCAGGCCCAGGGGG 48  706-735  CTGCCCCCGCGCCTCCTCGCACCCGGGGCT 49  721-750  AGACTTCGGCTGGCACTGCCCCCGCGCCTC 50  736-765  CTCTTGGGCAACGGCAGACTTCGGCTGGCA 51  751-780  GCGCCACGCCTGGGCCTCTTGGGCAACGGC 52  766-795  TCCGGCTCAGGGGCAGCGCCACGCCTGGGC 53  781-810  CCAACGGGCGTCCGCTCCGGCTCAGGGGCA 54  796-825  GCCCAGGACCCCTGCCCAACGGGCGTCCGC 62  916-945  GGGTGGGAGTGGCGCGTGCCAGAGAGCGCA 68 1006-1035 TCGGCGTACACCGGGGGACAAGGCGTGTCC 69 1021-1050 AGGAAGTGCTTGGTCTCGGCGTACACCGGG 70 1036-1065 TCGCCTGAGGAGTAGAGGAAGTGCTTGGTC 71 1051-1080 CGCAGCTGCTCCTTGTCGCCTGAGGAGTAG 72 1066-1095 AGTAGGAAGGAGGGCCGCAGCTGCTCCTTG 73 1081-1110 GGCCTCAGAGAGCTGAGTAGGAAGGAGGGC 74 1096-1125 GCGCCAGTCAGGCTGGGCCTCAGAGAGCTG 75 1111-1140 TCCACGAGCCTCCGAGCGCCAGTCAGGCTG 76 1126-1155 CCCAGAAAGATGGTCTCCACGAGCCTCCGA 77 1141-1170 ATCCAGGGCCTGGAACCCAGAAAGATGGTC 80 1186-1215 CAGTAGCGCTGGGGCAGGCGGGGCAACCTG 81 1201-1230 AGGGGCCGCATTTGCCAGTAGCGCTGGGGC 82 1216-1245 AGCAGCTCCAGAAACAGGGGCCGCATTTGC 83 1231-1260 TGCGCGTGGTTCCCAAGCAGCTCCAGAAAC 84 1246-1275 ACCCCGTAGGGGCACTGCGCGTGGTTCCCA 85 1261-1290 TGCGTCTTGAGGAGCACCCCGTAGGGGCAC 86 1276-1305 GCTCGCAGCGGGCAGTGCGTCTTGAGGAGC 87 1291-1320 GCTGGGGTGACCGCAGCTCGCAGCGGGCAG 88 1306-1335 GCACAGACACCGGCTGCTGGGGTGACCGCA 93 1381-1410 AGCAGCTGCACCAGGCGACGGGGGTCTGTG 94 1396-1425 CTGCTGTGCTGGCGGAGCAGCTGCACCAGG 96 1426-1455 GCCCGCACGAAGCCGTACACCTGCCAGGGG 100 1486-1515 AAGCGGCGTTCGTTGTGCCTGGAGCCCCAG 112 1666-1695 CAGTGCAGGAACTTGGCCAGGATCTCCTCA 114 1696-1725 AGCAGCTCGACGACGTACACACTCATCAGC 130 1936-1965 TCCATGTTCACAATCGGCCGCAGCCCGTCA 143 2131-2160 GGGTCCTGGGCCCGCACACGCAGCACGAAG 144 2146-2175 TACAGCTCAGGCGGCGGGTCCTGGGCCCGC 151 2251-2280 CGCACGCAGTACGTGTTCTGGGGTTTGATG 152 2266-2295 ACCACGGCATACCGACGCACGCAGTACGTG 201 3001-3030 TTCACCTGCAAATCCAGAAACAGGCTGTGA 202 3016-3045 ACCGTCTGGAGGCTGTTCACCTGCAAATCC 203 3031-3060 TAGATGTTGGTGCACACCGTCTGGAGGCTG 208 3106-3135 TTCCAAACTTGCTGATGAAATGGGAGCTGC 209 3121-3150 AAAAATGTGGGGTTCTTCCAAACTTGCTGA 210 3136-3165 GAGATGACGCGCAGGAAAAATGTGGGGTTC 211 3151-3180 AGGGAGGCCGTGTCAGAGATGACGCGCAGG 212 3166-3195 AGGATGGAGTAGCAGAGGGAGGCCGTGTCA 213 3181-3210 GCGTTCTTGGCTTTCAGGATGGAGTAGCAG 230 3436-3465 GCGGGTGGCCATCAGTCCAGGATGGTCTTG 237 3541-3570 CAGACTCCCAGCGGTGCGGGCCTGGGTGTG 241 3601-3630 AGCCGGACACTCAGCCTTCAGCCGGACATG

[0065] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0066] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

EXAMPLES

[0067] The following examples are provided by way of illustration only and not by ion. Those of skill will readily recognize a variety of noncritical parameters changed or modified to yield essentially similar results.

[0068] Example I: Inhibition of hTRT expression in vitro

[0069] In this example, inhibition of hTRT expression was examined using an in vitro cell-free expression system. A series of 30-mer antisense phosphorothioate oligonucleotides (PS-ODNs), which span the entire hTRT sequence, was systematically assayed for the ability to block hTRT expression in vitro (see FIG. 3). Co-expression of luciferase was used to normalize the samples and demonstrate the specificity of inhibition.

[0070] For inhibition of hTRT expression in vitro, an hTRT transcription/expression plasmid was prepared according to standard methodology for in vitro transcription and translation of hTRT RNA. Coupled transcription-translation reactions were performed with a reticulocyte lysate system (Promega TNT™) according to standard conditions (as performed in Example 7, U.S. patent application Ser. No. 08/974,549). Each coupled transcription/translation reaction included hTRT RNA transcribed from the expression plasmid, and a test antisense polynucleotide at a range of standard test concentrations, as well as the luciferase transcription/translation internal control (see, e.g., Sambrook et al., supra, Ausubel et al., supra). The translation reaction can also be performed with hTRT RNA that is synthesized in vitro in a separate reaction and then added to the translation reaction. ³⁵S-Met was included in the reaction to label the translation products. The negative control was performed without added PS-ODN.

[0071] The labeled translation products were separated by gel electrophoresis and quantitated after exposing the gel to a phosphorimager screen. The amount of hTRT protein expressed in the presence of hTRT specific PS-ODNs was normalized to the co-expressed luciferase control. The data are presented in FIG. 3 as a percentage of the control, which is without added PS-ODN.

[0072] Example II: Inhibition of hTRT expression ex vivo

[0073] A. Reagents

[0074] Cells: ACHN cells, NCI, catalogue #503755; 293 cells, ATCC; BJ (see, e.g., Kim et al., Science 266: 2011-2015 (1994)); additional cells from the ATCC or NCI.

[0075] Media and solutions: RPMI 1640 medium, BioWhitaker; DMEM/M199 medium, BioWhitaker; EMEM, BioWhitaker; Fetal Bovine Serum, Summit (stored frozen at −20° C., stored thawed at 4° C.); Trypsin-EDTA, GIBCO (catalogue #25300-054) (stored frozen at-20° C., stored thawed 4° C.; Isoton II (stored at RT); DMSO (stored at RT); oligonucleotides (see Table 1 and FIG. 3, stored in solution at −20° C.); PBS (Ca⁺⁺/Mg⁺⁺free); TE; 10 mM Tris-HCL, pH 8.0; 1 mM EDTA.

[0076] To prepare oligonucleotide stocks: Dissolve oligonucleotide nucleotides (PS-ODNs) in the appropriate amount of TE to make a concentrated stock solution (1-20 mM).

[0077] B. Treatment of cells ex vivo with antisense hTRT oligonucleotides

[0078] 1. For plating cells prior to oligonucleotide treatment, stock cultures of cells in log-phase growth (in T75 flask) were used. ACNH, 293, and BJ cells were used in this assay. The media was removed by aspiration, and the cells were rinsed with 2-5 ml of PBS. 1 ml of trypsin-EDTA was added to the cells, swirled to distribute, and incubated for 2 minutes. The trypsin was inactivated with 9 ml of media. The cells were gently triturated with media. 200 μL of the cells were then counted with a Coulter counter and diluted to the appropriate volume and number of cells per well.

[0079] 2. For 6-well dishes, 1.1×10⁵ cells total per well, 2 ml/well were added. The cells were allowed to settle 4-6 h prior to any treatment with oligonucleotides. The amount of cells can be scaled up or down proportionally for 12-well, 100 mm, or 150 mm dishes. For example, for 12-well dishes, use 4.6 x 10⁴ cells in 2 ml media; for 100 mm dishes use 6×10⁵ cells in 10 ml media; for 150 mm dishes use 1.7×10⁶ cells in 35 ml media.

[0080] 3. Oligonucleotides were diluted in media and fed to the cells at a range of standard test concentrations. Serial, sterile dilutions of the ODNs (see, e.g., Table 1) were prepared in sterile, filtered media for feeding the cells. The cells were treated in single, duplicate, or triplicate wells. Control wells were treated with TE diluted in media.

[0081] 4. The cells were fed daily with freshly diluted PS-ODN-media by aspirating the media and then feeding with 2 ml of freshly diluted oligonucleotide in media.

[0082] 5. When cells were near 70-80% confluent (3-4 days), the number of cells was determined per well. The media was removed by aspiration, and the cells were rinsed twice with 2 ml PBS. 0.5 ml trypsin-EDTA was added to the cells, swirled, and incubated for 2 minutes. The cells were triturated gently with 2 ml media per well. 200 μL of cells were counted in a Coulter counter. If necessary, the cells are replated at 1.1×10⁵ cells per well, 2 ml media per well, and fed with PS-ODN as described above.

[0083] 6. Samples of the cells were also harvested for analysis of telomerase activity by TRAP activity. The cells can also be analyzed by isolating RNA and performing RT-PCR, by TRF measurement, or by telomere length measurement (see, e.g., Example section, U.S. patent application Ser. No. 08/974,549 for assay protocols).

[0084] 7. The cell population doublings (PDLS) were calculated for each timepoint according to the following formula. PDLs (P): Pn Pn−1+[((ln(Total # cells))-(ln(# cells plated))/1n(2)]

[0085] 8. Graph PDL vs. time (in days) for the full dose range of each PS-ODN as compared to control untreated cells.

[0086] 9. Steps 2-8 were repeated for the desired duration (usually 2-4 weeks) or until cell growth was inhibited significantly.

[0087] 10. Table 1 shows exemplary oligonucleotides that were tested using this assay, and which inhibited telomerase expression and activity by approximately 50 % or more.

1 72 4015 base pairs nucleic acid single linear cDNA CDS 56..3454 /product= “human telomerase reverse transcriptase (hTRT)” 1 GCAGCGCTGC GTCCTGCTGC GCACGTGGGA AGCCCTGGCC CCGGCCACCC CCGCG ATG 58 Met 1 CCG CGC GCT CCC CGC TGC CGA GCC GTG CGC TCC CTG CTG CGC AGC CAC 106 Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser His 5 10 15 TAC CGC GAG GTG CTG CCG CTG GCC ACG TTC GTG CGG CGC CTG GGG CCC 154 Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly Pro 20 25 30 CAG GGC TGG CGG CTG GTG CAG CGC GGG GAC CCG GCG GCT TTC CGC GCG 202 Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg Ala 35 40 45 CTG GTG GCC CAG TGC CTG GTG TGC GTG CCC TGG GAC GCA CGG CCG CCC 250 Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro Pro 50 55 60 65 CCC GCC GCC CCC TCC TTC CGC CAG GTG TCC TGC CTG AAG GAG CTG GTG 298 Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu Val 70 75 80 GCC CGA GTG CTG CAG AGG CTG TGC GAG CGC GGC GCG AAG AAC GTG CTG 346 Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val Leu 85 90 95 GCC TTC GGC TTC GCG CTG CTG GAC GGG GCC CGC GGG GGC CCC CCC GAG 394 Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro Glu 100 105 110 GCC TTC ACC ACC AGC GTG CGC AGC TAC CTG CCC AAC ACG GTG ACC GAC 442 Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr Asp 115 120 125 GCA CTG CGG GGG AGC GGG GCG TGG GGG CTG CTG CTG CGC CGC GTG GGC 490 Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val Gly 130 135 140 145 GAC GAC GTG CTG GTT CAC CTG CTG GCA CGC TGC GCG CTC TTT GTG CTG 538 Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val Leu 150 155 160 GTG GCT CCC AGC TGC GCC TAC CAG GTG TGC GGG CCG CCG CTG TAC CAG 586 Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr Gln 165 170 175 CTC GGC GCT GCC ACT CAG GCC CGG CCC CCG CCA CAC GCT AGT GGA CCC 634 Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly Pro 180 185 190 CGA AGG CGT CTG GGA TGC GAA CGG GCC TGG AAC CAT AGC GTC AGG GAG 682 Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg Glu 195 200 205 GCC GGG GTC CCC CTG GGC CTG CCA GCC CCG GGT GCG AGG AGG CGC GGG 730 Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg Gly 210 215 220 225 GGC AGT GCC AGC CGA AGT CTG CCG TTG CCC AAG AGG CCC AGG CGT GGC 778 Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg Gly 230 235 240 GCT GCC CCT GAG CCG GAG CGG ACG CCC GTT GGG CAG GGG TCC TGG GCC 826 Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp Ala 245 250 255 CAC CCG GGC AGG ACG CGT GGA CCG AGT GAC CGT GGT TTC TGT GTG GTG 874 His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val Val 260 265 270 TCA CCT GCC AGA CCC GCC GAA GAA GCC ACC TCT TTG GAG GGT GCG CTC 922 Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala Leu 275 280 285 TCT GGC ACG CGC CAC TCC CAC CCA TCC GTG GGC CGC CAG CAC CAC GCG 970 Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His Ala 290 295 300 305 GGC CCC CCA TCC ACA TCG CGG CCA CCA CGT CCC TGG GAC ACG CCT TGT 1018 Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro Cys 310 315 320 CCC CCG GTG TAC GCC GAG ACC AAG CAC TTC CTC TAC TCC TCA GGC GAC 1066 Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly Asp 325 330 335 AAG GAG CAG CTG CGG CCC TCC TTC CTA CTC AGC TCT CTG AGG CCC AGC 1114 Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro Ser 340 345 350 CTG ACT GGC GCT CGG AGG CTC GTG GAG ACC ATC TTT CTG GGT TCC AGG 1162 Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser Arg 355 360 365 CCC TGG ATG CCA GGG ACT CCC CGC AGG TTG CCC CGC CTG CCC CAG CGC 1210 Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln Arg 370 375 380 385 TAC TGG CAA ATG CGG CCC CTG TTT CTG GAG CTG CTT GGG AAC CAC GCG 1258 Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His Ala 390 395 400 CAG TGC CCC TAC GGG GTG CTC CTC AAG ACG CAC TGC CCG CTG CGA GCT 1306 Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg Ala 405 410 415 GCG GTC ACC CCA GCA GCC GGT GTC TGT GCC CGG GAG AAG CCC CAG GGC 1354 Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln Gly 420 425 430 TCT GTG GCG GCC CCC GAG GAG GAG GAC ACA GAC CCC CGT CGC CTG GTG 1402 Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu Val 435 440 445 CAG CTG CTC CGC CAG CAC AGC AGC CCC TGG CAG GTG TAC GGC TTC GTG 1450 Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe Val 450 455 460 465 CGG GCC TGC CTG CGC CGG CTG GTG CCC CCA GGC CTC TGG GGC TCC AGG 1498 Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser Arg 470 475 480 CAC AAC GAA CGC CGC TTC CTC AGG AAC ACC AAG AAG TTC ATC TCC CTG 1546 His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser Leu 485 490 495 GGG AAG CAT GCC AAG CTC TCG CTG CAG GAG CTG ACG TGG AAG ATG AGC 1594 Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met Ser 500 505 510 GTG CGG GAC TGC GCT TGG CTG CGC AGG AGC CCA GGG GTT GGC TGT GTT 1642 Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys Val 515 520 525 CCG GCC GCA GAG CAC CGT CTG CGT GAG GAG ATC CTG GCC AAG TTC CTG 1690 Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe Leu 530 535 540 545 CAC TGG CTG ATG AGT GTG TAC GTC GTC GAG CTG CTC AGG TCT TTC TTT 1738 His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe Phe 550 555 560 TAT GTC ACG GAG ACC ACG TTT CAA AAG AAC AGG CTC TTT TTC TAC CGG 1786 Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr Arg 565 570 575 AAG AGT GTC TGG AGC AAG TTG CAA AGC ATT GGA ATC AGA CAG CAC TTG 1834 Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His Leu 580 585 590 AAG AGG GTG CAG CTG CGG GAG CTG TCG GAA GCA GAG GTC AGG CAG CAT 1882 Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln His 595 600 605 CGG GAA GCC AGG CCC GCC CTG CTG ACG TCC AGA CTC CGC TTC ATC CCC 1930 Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro 610 615 620 625 AAG CCT GAC GGG CTG CGG CCG ATT GTG AAC ATG GAC TAC GTC GTG GGA 1978 Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val Gly 630 635 640 GCC AGA ACG TTC CGC AGA GAA AAG AGG GCC GAG CGT CTC ACC TCG AGG 2026 Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser Arg 645 650 655 GTG AAG GCA CTG TTC AGC GTG CTC AAC TAC GAG CGG GCG CGG CGC CCC 2074 Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg Pro 660 665 670 GGC CTC CTG GGC GCC TCT GTG CTG GGC CTG GAC GAT ATC CAC AGG GCC 2122 Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg Ala 675 680 685 TGG CGC ACC TTC GTG CTG CGT GTG CGG GCC CAG GAC CCG CCG CCT GAG 2170 Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro Glu 690 695 700 705 CTG TAC TTT GTC AAG GTG GAT GTG ACG GGC GCG TAC GAC ACC ATC CCC 2218 Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile Pro 710 715 720 CAG GAC AGG CTC ACG GAG GTC ATC GCC AGC ATC ATC AAA CCC CAG AAC 2266 Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln Asn 725 730 735 ACG TAC TGC GTG CGT CGG TAT GCC GTG GTC CAG AAG GCC GCC CAT GGG 2314 Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His Gly 740 745 750 CAC GTC CGC AAG GCC TTC AAG AGC CAC GTC TCT ACC TTG ACA GAC CTC 2362 His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp Leu 755 760 765 CAG CCG TAC ATG CGA CAG TTC GTG GCT CAC CTG CAG GAG ACC AGC CCG 2410 Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser Pro 770 775 780 785 CTG AGG GAT GCC GTC GTC ATC GAG CAG AGC TCC TCC CTG AAT GAG GCC 2458 Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu Ala 790 795 800 AGC AGT GGC CTC TTC GAC GTC TTC CTA CGC TTC ATG TGC CAC CAC GCC 2506 Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His Ala 805 810 815 GTG CGC ATC AGG GGC AAG TCC TAC GTC CAG TGC CAG GGG ATC CCG CAG 2554 Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro Gln 820 825 830 GGC TCC ATC CTC TCC ACG CTG CTC TGC AGC CTG TGC TAC GGC GAC ATG 2602 Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp Met 835 840 845 GAG AAC AAG CTG TTT GCG GGG ATT CGG CGG GAC GGG CTG CTC CTG CGT 2650 Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu Arg 850 855 860 865 TTG GTG GAT GAT TTC TTG TTG GTG ACA CCT CAC CTC ACC CAC GCG AAA 2698 Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala Lys 870 875 880 ACC TTC CTC AGG ACC CTG GTC CGA GGT GTC CCT GAG TAT GGC TGC GTG 2746 Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys Val 885 890 895 GTG AAC TTG CGG AAG ACA GTG GTG AAC TTC CCT GTA GAA GAC GAG GCC 2794 Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu Ala 900 905 910 CTG GGT GGC ACG GCT TTT GTT CAG ATG CCG GCC CAC GGC CTA TTC CCC 2842 Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe Pro 915 920 925 TGG TGC GGC CTG CTG CTG GAT ACC CGG ACC CTG GAG GTG CAG AGC GAC 2890 Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser Asp 930 935 940 945 TAC TCC AGC TAT GCC CGG ACC TCC ATC AGA GCC AGT CTC ACC TTC AAC 2938 Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe Asn 950 955 960 CGC GGC TTC AAG GCT GGG AGG AAC ATG CGT CGC AAA CTC TTT GGG GTC 2986 Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly Val 965 970 975 TTG CGG CTG AAG TGT CAC AGC CTG TTT CTG GAT TTG CAG GTG AAC AGC 3034 Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn Ser 980 985 990 CTC CAG ACG GTG TGC ACC AAC ATC TAC AAG ATC CTC CTG CTG CAG GCG 3082 Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln Ala 995 1000 1005 TAC AGG TTT CAC GCA TGT GTG CTG CAG CTC CCA TTT CAT CAG CAA GTT 3130 Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln Val 1010 1015 1020 1025 TGG AAG AAC CCC ACA TTT TTC CTG CGC GTC ATC TCT GAC ACG GCC TCC 3178 Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala Ser 1030 1035 1040 CTC TGC TAC TCC ATC CTG AAA GCC AAG AAC GCA GGG ATG TCG CTG GGG 3226 Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu Gly 1045 1050 1055 GCC AAG GGC GCC GCC GGC CCT CTG CCC TCC GAG GCC GTG CAG TGG CTG 3274 Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp Leu 1060 1065 1070 TGC CAC CAA GCA TTC CTG CTC AAG CTG ACT CGA CAC CGT GTC ACC TAC 3322 Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr Tyr 1075 1080 1085 GTG CCA CTC CTG GGG TCA CTC AGG ACA GCC CAG ACG CAG CTG AGT CGG 3370 Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser Arg 1090 1095 1100 1105 AAG CTC CCG GGG ACG ACG CTG ACT GCC CTG GAG GCC GCA GCC AAC CCG 3418 Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn Pro 1110 1115 1120 GCA CTG CCC TCA GAC TTC AAG ACC ATC CTG GAC TGATGGCCAC CCGCCCAC 3471 Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 CCAGGCCGAG AGCAGACACC AGCAGCCCTG TCACGCCGGG CTCTACGTCC CAGGGAGG 3531 GGGGCGGCCC ACACCCAGGC CCGCACCGCT GGGAGTCTGA GGCCTGAGTG AGTGTTTG 3591 CGAGGCCTGC ATGTCCGGCT GAAGGCTGAG TGTCCGGCTG AGGCCTGAGC GAGTGTCC 3651 CCAAGGGCTG AGTGTCCAGC ACACCTGCCG TCTTCACTTC CCCACAGGCT GGCGCTCG 3711 TCCACCCCAG GGCCAGCTTT TCCTCACCAG GAGCCCGGCT TCCACTCCCC ACATAGGA 3771 AGTCCATCCC CAGATTCGCC ATTGTTCACC CCTCGCCCTG CCCTCCTTTG CCTTCCAC 3831 CCACCATCCA GGTGGAGACC CTGAGAAGGA CCCTGGGAGC TCTGGGAATT TGGAGTGA 3891 AAAGGTGTGC CCTGTACACA GGCGAGGACC CTGCACCTGG ATGGGGGTCC CTGTGGGT 3951 AATTGGGGGG AGGTGCTGTG GGAGTAAAAT ACTGAATATA TGAGTTTTTC AGTTTTGA 4011 AAAA 4015 1132 amino acids amino acid linear protein 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 3 TCCCACGTGC GCAGCAGGAC GCAGCGCTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 4 GGCATCGCGG GGGTGGCCGG GGCCAGGGCT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 5 CAGCGGGGAG CGCGCGGCAT CGCGGGGGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 6 AGCACCTCGC GGTAGTGGCT GCGCAGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 7 AACGTGGCCA GCGGCAGCAC CTCGCGGTAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 8 GCGGGGGGCG GCCGTGCGTC CCAGGGCACG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 9 CCGCGCTCGC ACAGCCTCTG CAGCACTCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 10 GGGGGGCCCC CGCGGGCCCC GTCCAGCAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 11 GTGGTGAAGG CCTCGGGGGG GCCCCCGCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 12 TAGCTGCGCA CGCTGGTGGT GAAGGCCTCG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 13 ACCGTGTTGG GCAGGTAGCT GCGCACGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 14 CGCAGTGCGT CGGTCACCGT GTTGGGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 15 AGGTGAACCA GCACGTCGTC GCCCACGCGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 16 GGGGGCCGGG CCTGAGTGGC AGCGCCGAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 17 CCACTAGCGT GTGGCGGGGG CCGGGCCTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 18 GCCCGTTCGC ATCCCAGACG CCTTCGGGGT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 19 ACGCTATGGT TCCAGGCCCG TTCGCATCCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 20 ACCCCGGCCT CCCTGACGCT ATGGTTCCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 21 GGCAGGCCCA GGGGGACCCC GGCCTCCCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 22 CTCGCACCCG GGGCTGGCAG GCCCAGGGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 23 CTGCCCCCGC GCCTCCTCGC ACCCGGGGCT 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 24 AGACTTCGGC TGGCACTGCC CCCGCGCCTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 25 CTCTTGGGCA ACGGCAGACT TCGGCTGGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 26 GCGCCACGCC TGGGCCTCTT GGGCAACGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 27 TCCGGCTCAG GGGCAGCGCC ACGCCTGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 28 CCAACGGGCG TCCGCTCCGG CTCAGGGGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 29 GCCCAGGACC CCTGCCCAAC GGGCGTCCGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 30 GGGTGGGAGT GGCGCGTGCC AGAGAGCGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 31 TCGGCGTACA CCGGGGGACA AGGCGTGTCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 32 AGGAAGTGCT TGGTCTCGGC GTACACCGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 33 TCGCCTGAGG AGTAGAGGAA GTGCTTGGTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 34 CGCAGCTGCT CCTTGTCGCC TGAGGAGTAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 35 AGTAGGAAGG AGGGCCGCAG CTGCTCCTTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 36 GGCCTCAGAG AGCTGAGTAG GAAGGAGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 37 GCGCCAGTCA GGCTGGGCCT CAGAGAGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 38 TCCACGAGCC TCCGAGCGCC AGTCAGGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 39 CCCAGAAAGA TGGTCTCCAC GAGCCTCCGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 40 ATCCAGGGCC TGGAACCCAG AAAGATGGTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 41 CAGTAGCGCT GGGGCAGGCG GGGCAACCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 42 AGGGGCCGCA TTTGCCAGTA GCGCTGGGGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 43 AGCAGCTCCA GAAACAGGGG CCGCATTTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 44 TGCGCGTGGT TCCCAAGCAG CTCCAGAAAC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 45 ACCCCGTAGG GGCACTGCGC GTGGTTCCCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 46 TGCGTCTTGA GGAGCACCCC GTAGGGGCAC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 47 GCTCGCAGCG GGCAGTGCGT CTTGAGGAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 48 GCTGGGGTGA CCGCAGCTCG CAGCGGGCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 49 GCACAGACAC CGGCTGCTGG GGTGACCGCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 50 AGCAGCTGCA CCAGGCGACG GGGGTCTGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 51 CTGCTGTGCT GGCGGAGCAG CTGCACCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 52 GCCCGCACGA AGCCGTACAC CTGCCAGGGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 53 AAGCGGCGTT CGTTGTGCCT GGAGCCCCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 54 CAGTGCAGGA ACTTGGCCAG GATCTCCTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 55 AGCAGCTCGA CGACGTACAC ACTCATCAGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 56 TCCATGTTCA CAATCGGCCG CAGCCCGTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 57 GGGTCCTGGG CCCGCACACG CAGCACGAAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 58 TACAGCTCAG GCGGCGGGTC CTGGGCCCGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 59 CGCACGCAGT ACGTGTTCTG GGGTTTGATG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 60 ACCACGGCAT ACCGACGCAC GCAGTACGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 61 TTCACCTGCA AATCCAGAAA CAGGCTGTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 62 ACCGTCTGGA GGCTGTTCAC CTGCAAATCC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 63 TAGATGTTGG TGCACACCGT CTGGAGGCTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 64 TTCCAAACTT GCTGATGAAA TGGGAGCTGC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 65 AAAAATGTGG GGTTCTTCCA AACTTGCTGA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 66 GAGATGACGC GCAGGAAAAA TGTGGGGTTC 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 67 AGGGAGGCCG TGTCAGAGAT GACGCGCAGG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 68 AGGATGGAGT AGCAGAGGGA GGCCGTGTCA 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 69 GCGTTCTTGG CTTTCAGGAT GGAGTAGCAG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 70 GCGGGTGGCC ATCAGTCCAG GATGGTCTTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 71 CAGACTCCCA GCGGTGCGGG CCTGGGTGTG 30 30 base pairs nucleic acid single linear other nucleic acid /desc = “phosphorothioate oligonucleotide” 72 AGCCGGACAC TCAGCCTTCA GCCGGACATG 30 

What is claimed is:
 1. A method of inhibiting telomerase expression in a cell or growth of a cell, comprising introducing into the cell an isolated antisense oligonucleotide that specifically hybridizes to an hTRT polynucleotide comprising the nucleotide sequence SEQ. ID NO:1.
 2. The method of claim 1, whereby the oligonucleotide reduces telomerase expression in the cell by at least 50%.
 3. The method of claim 1, whereby the oligonucleotide inhibits telomerase catalytic activity in the cell.
 4. The method of claim 1, whereby the oligonucleotide reduces replicative capacity of the cell.
 5. The method of claim 1, whereby the oligonucleotide causes cleavage of hTRT mRNA in the cell.
 6. The method of claim 1, wherein the oligonucleotide is DNA.
 7. The method of claim 1, wherein the oligonucleotide is RNA.
 8. The method of claim 1, wherein the oligonucleotide is a ribozyme.
 9. The method of claim 1, wherein the oligonucleotide comprises one or more non-naturally occurring nucleotides.
 10. The method of claim 1, wherein the oligonucleotide comprises a phosphorothioate nucleotide.
 11. The method of claim 1, wherein the oligonucleotide is from ˜10 to 50 nucleotides in length.
 12. The method of claim 1, wherein the oligonucleotide is from ˜20 to 100 nucleotides in length.
 13. The method of claim 1, wherein the oligonucleotide comprises a sequence of ˜7 to 100 nucleotides that is exactly complementary to a sequence contained in SEQ. ID NO:1.
 14. The method of claim 1, wherein the oligonucleotide comprises two sequences of ˜7 to 100 nucleotides that are complementary to sequences contained in SEQ. ID NO: 1, flanking a ribozyme catalytic site.
 15. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ. ID NOs:4-72.
 16. The oligonucleotide of claim 1, consisting essentially of ˜7 to 100 consecutive nucleotides complementary to a sequence contained in SEQ. ID NO: 1, except for one or more silent variations.
 17. The oligonucleotide of claim 1, which inhibits hTRT expression in cultured cells by at least 75%.
 18. The oligonucleotide of claim 1, which inhibits hTRT expression in cultured cells by at least 95%. 